The long-term goals of the research programs of both the PI (Visconti) and Foreign Collaborator (FC; Darszon) are to understand the molecular basis of the sperm ability to fertilize. Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire the capacity to fertilize after residing in the female tract for a finite period of time. The physiological changes occurring in the female reproductive tract, that render the sperm able to fertilize, constitute the phenomenon of "sperm capacitation". Work by the PI has focused on the signaling events controlling this poorly understood process. Work of the FC has focused on the study of ion channels and on the regulation of ionic events in mammalian and invertebrate sperm. Recently they described an inward rectifier K+ channel that contributes to the hyperpolarization associated to capacitation. Together the FC and Pi have demonstrated that a Na+/HCO3- cotransporter is present in mouse sperm and has a role in mouse sperm capacitation. This FIRCA application is based on observations made during these studies. The findings of the PI and FC indicate that the mouse sperm resting membrane potential (Era) has an important contribution of permeability towards Na+ and K+ ions. In the first aim, experiments will be carried out to determine how external Na+ influences sperm Em before and after capacitation and which Na+ transporters are involved. In the second aim Na+ channels will be measured in mouse spermatogenic cells by patch clamp techniques and in uncapacitated and capacitated sperm using planar bilayers. The presence of amiloride sensitive Na+ channels and hyperpolarization and cyclic nucleotide gated (HCN) channels in spermatogenic cells and sperm will be explored. Finally, in the third aim, experiments will be conducted to study the regulation of the inward rectifier K+ (Kir) channel present in mouse spermatogenic cells that participates in sperm capacitation. RT-PCR and immunological approaches will be used to establish the molecular identity of these Kir channels. Dr. Darszon is an internationally recognized expert in all the techniques proposed in the present application. A clear understanding of the molecular mechanisms of capacitation and the role of ions in capacitation is relevant to research in sperm based new targets for non hormonal contraceptive approaches as well as to investigation on improvements of the fertilization rates in infertile couples.